Methods for producing low binding surfaces

Description

FIELD OF THE INVENTION

[0001] This invention relates to methods for reducing the binding of organic materials (e.g., peptides, proteins, nucleic acids, and cells) to hydrophobic surfaces (e.g., polymeric surfaces). In particular, the invention relates to producing articles of manufacture (e.g., labware) having such low binding surfaces.

BACKGROUND OF THE INVENTION

[0002] Biological materials such as peptides, proteins, nucleic acids, and cells are often stored or transferred in containers such as centrifuge tubes and pipettes made of plastic or other hydrophobic materials. It is a common observation that biological compounds adsorb/bind to the surfaces of such containers. This is also true for organic materials which exhibit some hydrophobicity in an aqueous solution, e.g., acridinium compounds, PCBs, etc..

[0003] For many applications, such binding is undesirable. For example, the binding results in the loss of valuable materials, such as, enzymes and antibodies, and can result in variations in the dispensing of organic materials, especially when small volumes are involved. The binding of proteins, cells, and platelets to hydrophobic surfaces is also of concern in a variety of blood handling procedures.

[0004] As a result of these considerations, extensive efforts have been made to provide methods for reducing the binding of proteins and other organic compounds to hydrophobic surfaces. Examples of the approaches which have been considered can be found in Caldwell et al., U.S. Patent No. 5,516,703; Ding et al., International Application Publication WO94/03544; Amiji et al., Biomaterials, 13:682-692, 1992; J. Andrade, "Principles of Protein Adsorption" in Surface and Interfacial Aspects of Biomedical Polymers, J. Andrade, editor, Volume 2, Plenum Press, New York, 1-80, 1985; Lee et al., Polymeric Mater. Sci Eng., 57:613-617, 1987; Lee et al., Journal of Biomedical Materials Research, 23:351-368, 1989; Lee et al., Biomaterials, 11:455-464, 1990; Lee et al., Prog. Polym. Sci., 20:1043-1079, 1995; Merrill et al., ASAIO Journal, 6:60-64, 1983; Okano et al., Journal of Biomedical Materials Research, 20:1035-1047, 1986; Okkema et al., J. Biomater. Sci. Polymer Edn., 1:43-62, 1989; Owens et al., Journal of Cell Science, 87:667-675, 1987; Rabinow et al., J. Biomater. Sci. Polymer Edn., 6:91-109, 1994; Schroën et al., Journal of Membrane Science, 80:265-274, 1993; Sheu et al., J. Adhesion Sci. Technol., 6:995-1009, 1992; Shimada et al., Polymer Journal, 15:649-656, 1983; and Thurow et al., Diabetologia, 27:212-218, 1984.

[0005] The criteria which a successful technique for producing a low binding surface should satisfy include: 1) a sufficiently low level of binding; 2) substantial permanence; 3) ease of use; and 4) low cost. It is the goal of the present invention to provide methods for producing low binding surfaces which satisfy all of these criteria.

SUMMARY OF THE INVENTION

[0006] The present invention achieves the above criteria through the combination of specific coating materials and specific process steps, both of which are critical to the success of the technique.

[0007] The present invention provides a method for making a body composed of a hydrophobic polymer, said method comprising: (a) providing a mold for forming the body; (b) applying a non-ionic surfactant to a surface of the mold, said non-ionic surfactant having (i) a hydrophilic-lipophilic balance number which is less than or equal to 5 and (ii) at least one hydrophilic element which can extend into an aqueous solution; and (c) using the mold to form the body with at least part of the body being formed by contact with the surface to which the non-ionic surfactant was applied so that at least some non-ionic surfactant is transferred from the surface to said part of the body.

[0008] The specific materials employed in the invention are non-ionic surfactants which have a hydrophilic element which can extend into an aqueous solution, e.g., a hydrophilic end group, and have a hydrophilic-lipophilic balance number (HLB number) which is less than or equal to 5. The term "non-ionic surfactant" is used herein in accordance with its classical definition as a molecule containing two structurally dissimilar groups having different solubilities in an aqueous solution. See Kirk-Othmer Encyclopedia of ChemicalTechnology, Third Edition, Volume 22, page 332, John Wiley & Sons, New York, New York, 1983.

[0009] As demonstrated in the examples presented below, a HLB number less than or equal to 5 has been found critical to achieve a durable low binding surface. Although non-ionic surfactants have been previously considered for use in producing low-binding surfaces (see the references cited above), the criticality of a HLB number less than or equal to 5 has not previously been recognized. As the present invention demonstrates, above this number, protein binding is either not substantially inhibited or is only temporally inhibited, while at or below the number, long term inhibition of protein binding is achieved.

[0010] One specific process described in EP 0936951, from which this application is divided, comprises the steps of applying the non-ionic surfactant to the surface (substrate) in a solvent and then drying the surface (substrate) to remove the solvent and thereby bring the surfactant into direct contact with the surface so as to bind thereto. Preferably, the surface is fully dried. The applying and drying steps must be performed without an intermediate washing step with an organic solvent.

[0011] In accordance with the invention, a surfactant having the characteristics described above, i.e., a HLB number less than or equal to 5 and a hydrophilic element which can extend into an aqueous solution, is applied to the mold used to make the part by, for example, spraying a solution of the surfactant onto at least one of the mold's molding surfaces. In accordance with the invention, it has been found that when such a treated mold is used to make parts, a sufficient amount of surfactant is transferred from the mold to the surface of the part so.as to produce a lowbinding surface. Although the mold can be sprayed with the surfactant each time a part is made, less frequent spraying can be used if desired. As with the post formation procedures described above, these as-the-part-is-made procedures satisfy all of the criteria for a practical process for producing a low-binding surface.

[0012] The process is inexpensive since only very low concentrations of surfactant are needed to achieve a low-binding surface. For example, one pound of surfactant which costs about a dollar (U.S.), can provide a micron thick coating on about 5,000 square feet (465 square meters) of hydrophobic surface. The invention thus satisfies each of the above four criteria for a practical process for producing a low-binding surface, i.e., it provides a low cost, easy-to-use procedure for providing a substantially permanent, low binding surface.

[0013] The process of the invention is even simpler and less expensive than the process described in EP 0936951 because the low-binding surface is created at the same time the part which is to have such a surface is formed.

[0014] Those references which have employed non-ionic surfactants having HLB numbers less than or equal to 5 have not disclosed, suggested, or in any way recognized the criticality of the above process steps. Specifically, the Thurow et al. and Schroën et al. references cited above each use at least one non-ionic surfactant having a HLB number less than 5. In particular, Thurow et al.'s preferred Genapol PF-10 material has a HLB number of less than 5, as does Schroën et al.'s L-92 material. While Thurow et al report that Genapol PF-10 prevents adsorption of insulin to latex particles, Schoën et al. report that L-92 does not prevent adsorption of lipase to a polypropylene membrane. Significantly, neither reference describes drying the non-ionic surfactant onto a hydrophobic surface, and thus neither can produce a low-binding surface which is substantially permanent, as is required for a practical product.

[0015] Sheu et al. also use non-ionic surfactants having low HLB numbers (i.e., PLURONIC 121, 122, and 127), but employ a complicated argon glow discharge process to bind these surfactants to a hydrophobic surface, namely, low density polyethylene (LDPE). In certain experiments, they omitted the glow discharge treatment and instead merely applied the surfactants to LDPE and washed with chloroform (see their Figure 2).

[0016] Under these conditions, they reported no reduction in protein binding compared to untreated LDPE (see their page 1006). Given this conclusion, Sheu et al. clearly did not recognize that low HLB surfactants could be successfully used to produce low-binding surfaces without the need for glow discharge treatment, as demonstrated by the present invention.

[0017] A particularly advantageous application of the invention is in the production of labware having protein resistant surfaces. Examples of the types of products which can be provided with low-binding surfaces in accordance with the invention include containers of all shapes, sizes, and descriptions, multiwell strips, pipettes, pipette tips, membranes, reagent reservoirs, storage vessels, tubing and the like. Once provided with a low binding surface, these products can be sterilized using conventional techniques such as gamma-ray sterilization.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] As discussed above, the present invention relates to the creation of low-binding surfaces on hydrophobic substrates through the use of nonionic surfactants having a HLB number less than or equal to 5 and a hydrophilic element which can extend into an aqueous solution. Discussions of HLB numbers and how they are determined for specific surfactants can be found in, for example, the publication of ICI Surfactants entitled The HLB System and, in particular, in Chapter 7 of that publication entitled "How to Determine HLB of an Emulsifier" (ICI Americas, Inc., Wilmington, Delaware, 1992).

[0019] The non-ionic surfactant used in the practice of the invention needs to have a hydrophilic element which can extend inton aqueous solution so as to provide the requisite low-binding surface. Although not wishing to be bound by any particular theory of operation, it is believed that such a hydrophilic element when hydrated and extending away from a hydrophobic surface provides an aqueous boundary layer which cannot be readily penetrated by molecules having hydrophobic regions, e.g., proteins, thus preventing such molecules from binding to the hydrophobic surface. In many cases, the non-ionic surfactant molecules used in the practice of the invention will have a central hydrophobic region connected at each end to a hydrophilic element which can extend into an aqueous solution. In other cases, the molecules will have a hydrophobic region connected on only one end to a hydrophilic element. Surfactant molecules having other configurations can, of course, be used if desired provided they have at least one hydrophilic element which can extend into an aqueous solution. It should be noted that in the limit, the hydrophilic element can be as simple as a hydroxyl group as demonstrated by the low binding achieved with polyproylene oxide (see Example 6 below).

[0020] The presence of the hydrophilic element or elements means that the surfactant molecules will normally have a HLB number greater than zero, i.e., they will have some hydrophilic character. (Note that in the case of polypropylene oxide, the HLB number is in effect close to zero, i.e., it is less than 0.5.) However, since the HLB number must be less than or equal to 5 to achieve a substantially permanent, low-binding surface, this hydrophilic character is significantly less than the molecule's lipophilic character. Generally, non-ionic surfactants having HLB numbers less than about 2.5 are preferred for the practice of the invention.

[0021] A variety of non-ionic surfactants now known or subsequently developed can be used in the practice of the invention. Examples of suitable surfactants include alkyl alcohol ethyoxylates, alkyl ester ethyoxylates, sorbitol alkyl esters, glycerol alkyl esters, and ethylene oxide/propylene oxide block co-polymers. As discussed above, polypropylene oxide can also be used in the practice of the invention. Preferably, the polypropylene oxide will have an average molecular weight in the range of from about 1,000 to about 15,000 Derivatives of polypropylene oxide, such as branched and star polymers, can also be used in the practice of the invention.

[0022] These and other suitable surfactants can be obtained from a variety of manufacturers including ICI Americas, Inc., Wilmington, Delaware; BASF Corp., Parsippany, New Jersey; Witco Corp., Greenwich, Connecticut; and the Henkel Corporation Ambler, Pennsylvania. If desired, mixtures of non-ionic surfactants can be used in the practice of the invention, provided each surfactant used in the mixture has a HLB number less than 5. Lists of various commercially available non-ionic surfactants can be found in McCutcheon's Emulsifiers and Detergents, North American edition, The Manufacturing Confectioner Publishing Co., Glen Rock, New Jersey, 1995. A preferred non-ionic surfactant for use in the present invention is Pluronic® L-121.

[0023] A variety of hydrophobic bodies can be made low-binding in accordance with the invention. As used herein, a body is considered to have been made low binding if the body exhibits a level of binding of the organic material which is less than 0.5 times and preferably less than 0.3 times the level of binding exhibited by a body formed using a mold to which the non-ionic surfactant has not been applied. Similarly, a surface treatment is considered to be substantially permanent if the treated surface retains its low protein binding properties after at least about 2 water washes at room temperature and preferably after at least about 6 washes, again at room temperature, where a water wash as used herein lasts at least 60 seconds.

[0024] Examples of the types of polymeric bodies which can benefit from the invention include those comprising or composed of polystyrene, polypropylene, polymethyl methacrylate, polyvinyl chloride, polymethyl pentene, polyethylene, polycarbonate, polysulfone, polystyrene copolymers (e.g., SAN and ABS), polypropylene copolymers, fluoropolymers, polyamides, silicones, and elastomers, including silicone, hydrocarbon, and fluorocarbon elastomers. Other materials can be used provided they have a hydrophobic surface to which the surfactant can bind.

[0025] As discussed above, rather than being applied to finished parts, the present invention relates to non-ionic surfactants applied to the mold used to form the part. In accordance with these embodiments, all or a portion of the mold surface is sprayed with a solution containing the non-ionic surfactant, the mold is closed, molten polymer is injected into the mold and cooled, the mold open is opened, and the molded part is ejected from the mold.

[0026] The non-ionic surfactants are applied to the mold surface in the form of a coating solution comprising the surfactant and a solvent. In view of the surfactant's low HLB number, the solvent is typically an organic solvent, a mixture of organic solvents, or a mixture of water and one or more miscible organic solvents, e.g., a water/alcohol mixture. The solvent should be one that can be easily evaporated. Solvents which are primarily composed of water are not preferred, although they can be used if desired. Such solvents evaporate relatively slowly and can lead to agglomeration problems in view of the low HLB number of the surfactant. Also, when used to spray a mold, any water which has not evaporated by the time the mold is closed and molten polymer is injected, will likely cause defects in the finished part.

[0027] The concentration of the surfactant in the coating solution can vary quite widely depending upon the application. Convenient concentrations are in the range of from about 0.01% weight per volume to about 1.0% weight per volume. A suitable concentration for the post formation coating of labware is about 0.1% weight per volume. Higher or lower concentrations can, of course, be used if desired.

[0028] A small quantity of surfactant can be used to treat a large surface area. Accordingly, the volume of coating solution applied per square millimeter of surface can be quite small, e.g., about 2 to 20 microliters per cm² for a coating solution having a surfactant concentration of about 0.1% weight per volume. The amount of surfactant per unit area and the corresponding coating solution concentration and application rate can be readily determined for any particular application by examining test pieces of the hydrophobic surface to determine if the requisite reduction in binding has been achieved.

[0029] In accordance with the invention, it has been found that sufficient surfactant is transferred to the surface of the polymer to produce a substantially permanent, non-binding surface. Although spraying is preferred, other techniques, e.g. brush coating, can be used to apply the surfactant to the mold. Equipment of the type used to apply release agents to molds can be used to apply the surfactant.

[0030] Without intending to limit it in any manner, the present invention will be more fully described by the following examples in which Examples 1 to 14 are included for comparative purposes and Example 15 is in accordance with the invention. The materials and methods which are common to the examples are as follows.

Materials and Methods

[0031] Table 14 sets forth the non-ionic surfactants used in the examples along with their HLB numbers and their commercial tradenames where applicable.

[0032] Except where indicated, in Examples 1 to 14 the non-ionic surfactants were applied to the hydrophobic surfaces being tested as a 0.1 % (weight/volume) solution in isopropanol. A sufficient amount of coating solution was used to cover the surface being tested with a thin layer of the solution (e.g., 25 µl of the solution per well for a standard 24-well plate). In some cases, the solution was applied to surface by spraying; in others, it was poured onto the surface (into the well). Except where indicated, coated samples were either dried at 70°C for 30 minutes in an air circulated oven or dried at room temperature overnight to evaporate the isopropanol.

[0033] Protein binding was determined in some examples using a colloidal gold straining procedure. In accordance with this procedure, colloidal gold becomes electrostatically attached to bound protein and is detected by measuring absorbance at 550 nanometers. Bio-Rad Laboratories' Colloidal Gold Total Protein Stain was used for this purpose, with absorbance being measured with a Cambridge Technologies Plate Reader (No. 7520).

[0034] As generally understood in the art, the upper limit for a low binding surface is about 50-80 ng/cm². For comparison, medium binding, such as that exhibited by untreated polystyrene or polypropylene, is around 200-400 ng/cm², and high binding, such as that exhibited by a polystyrene or polypropylene surface which has been plasma oxidized and gamma radiation sterilized, is around 400-800 ng/cm².

[0035] The designations "NA" and "ND" used in some tables mean "not applicable" and "not determined," respectively. The designation "PS" means polystyrene. The designation "M.W." means average molecular weight.

Example 1

Radiometric Determination of Reduced Protein Binding

[0036] This example demonstrates by means of a radiometric readout that non-ionic surfactants can reduce the binding of protein to hydrophobic surfaces.

[0037] The hydrophobic surfaces used were flat bottom microtiter plates composed of high binding polystyrene (Corning Costar No. 2581) and medium binding polystyrene (Corning Costar No. 2587). Testing was performed with and without gamma radiation sterilization using cobalt 60 (1.5 Mrad).

[0038] The non-ionic surfactants used in this example are set forth in Table 1. The surfactants were applied to the test plates as described in Materials and Methods, followed by sterilization where indicated.

[0039] Levels of protein binding were determined using a radiolabeled protein, namely, ¹²⁵I-IgG (goat anti-mouse) purchased from DuPont/NEN. Unlabeled IgG in sodium carbonate buffer pH 9.2 was spiked with ¹²⁵I-IgG so that the final concentration of labeled IgG and cold IgG in the test solution was 10 µg/mL.

[0040] Aliquots of 0.1 milliliters of the protein solution were placed in quadruplicate in the wells of 8-well strips removed from the test microtiter plates. The wells were incubated overnight at 4°C with shaking. The supernatants were removed from the wells, followed by three washes with 0.2 mL of PBS. The wells were then dried and individually counted to determine "total" IgG binding. After determining "total" binding, the wells were incubated for two hours in 0.2 mL of PBS-Tween (0.05%) at room temperature with shaking. The supernatants were removed from the wells followed by three washes with 0.2 mL of PBS. The wells were dried and individually counted to determine "tight" IgG binding.

[0041] Untreated high binding and medium binding plates were used as controls. A comparative experiment was also performed in which a BSA protein coating was used in place of a surfactant coating. The BSA was preabsorbed onto the walls of the wells and then washed to remove excess material. Testing was then performed using the radiolabeled IgG solution as described above.

[0042] Radiolabel measurements were performed in a double blind study at BioMolecular Assays (Woburn, MA). IgG binding values per microwell were converted to ng/cm² values using a surface area of 0.94 cm² for a 0.1 mL volume.

[0043] As shown in Table 1, the non-ionic surfactants tested each resulted in a substantial decrease in protein binding in comparison to the two controls and the BSA experiment, with the binding after sterilization being greater in most, but not all, cases, but still significantly reduced from the controls.

Example 2

Enzymatic Determination of Reduced Protein Binding

[0044] This example demonstrates by means of an enzymatic readout that non-ionic surfactants can reduce the binding of protein to hydrophobic surfaces.

[0045] The non-ionic surfactants used in this example are set forth in Table 2. The surfactants were coated onto a medium binding polystyrene plate (Corning Costar No. 2587) as described in Material and Methods. A medium binding polystyrene plate (Corning Costar No. 2587) and a high binding polystyrene plate (Corning Costar No. 2581) were used as controls.

[0046] The reagents employed in this example were:

(a) HRP-Goat A'Mouse IgG: Kirkegaard & Perry Laboratories, Inc. Catalog No. 074-1806. Stock solution: 0.5 mg/ml; working concentration: 0.04 µg/ml. The working concentration was obtained by adding 11 µl of the stock solution per 25 mls of PBS pH 7.4 to produce a work stock (WS), and then diluting 1:6 (i.e., 10 mls WS + 50 mls PBS pH 7.4).

(b) Phosphate Buffered Saline: Sigma 1000-3. Buffer preparation: 2 packages PBS per 2 liters H₂O.

(c) Wash solution 20X Concentrate: Kipergaard & Perry Laboratories, Inc. Catalog No. 50-63-01. Wash concentration: 1 M (3800 mls H₂O + 200 mls wash solution concentrate).

(d) ABTS Peroxidase Substrate & Peroxidase Solution B: Kirkegaard and Perry Laboratories, Inc. Catalog Nos. 50-64-02 & 50-65-02.

(e) Sodium Lauryl Sulfate (SDS): Sigma Catalog No. L-4509. Stop solution: 1% SDS (10 gm SDS per 1 liter H₂O).

[0047] The procedures employed were:

(1) 100 µl/well-of-HRP-Goa A'Mouse IgG at a concentration of 0.04 µg/ml were added to the test wells and incubated for 1 hour at room temperature.

(2) The wells were then washed 5 times with the 1M wash solution. Each wash include a 5 minute soak followed by decanting.

(3) 100 µl/well of substrate (30 mls ABTS + 30 mls H₂O₂) were added to the test wells and incubated for 15 minutes at room temperature in the dark.

(4) 100 µl/well of stopping solution (1% SDS) were added to the test wells.

(5) The plates were then shaken and read at 405 nm.

(6) Reduction in binding was determined by comparing the absorbance measured in step (5) with the absorbance measured for a high binding polystyrene plate which had not been treated with a non-ionic surfactant but otherwise subjected to steps (1) to (5).

[0048] As shown in Table 2 significant reductions in bound enzyme were achieved by the non-ionic surfactants, with most reductions being greater than 95%.

Example 3

Determination of Reduced Protein Binding by Colloidal Gold Staining

[0049] This example demonstrates by means of a colloidal gold readout that non-ionic surfactants can reduce the binding of protein to hydrophobic surfaces

[0050] The non-ionic surfactants used in this example are set forth in Table 3. The surfactants were coated onto medium binding polystyrene plates (Corning Costar No. 2587) as described in Material and Methods.

[0051] The plates were incubated with IgG as follows: 0.1 ml of Horse IgG (Pierce Labs) at a working concentration of 10µg/ml in 0.10M NaHCO₃ buffer (pH9.4) were added to each well, incubated 30 minutes on a rocker table, and rinsed with dI H₂O. The plates were gold stained overnight (0.3ml/well), rinsed in dI H₂O, air dried, and absorbance was measured at 550nm

[0052] As shown in Table 3, the non-ionic surfactants clearly achieved low protein binding levels, i.e., undetectable levels by the colloidal gold staining procedure.

Example 4

The Reduction in Protein Binding is pH Independent

[0053] This example demonstrates that non-ionic surfactants can inhibit protein binding over a wide range of pH's.

[0054] The non-ionic surfactants used in this example are set forth in Table 4. Polystyrene plates having 24 wells (Corning Costar # 9447) were coated as described in Materials and Methods.

[0055] BSA protein (Bovine Serum Albumin, Fraction V, Sigma) was prepared at 10 µg/ml in 0.10 M acetate buffer (pH=4.6), in 0.10 M PBS (pH=7.4), and in 0.10 M NaHCO₃ buffer (pH=9.2). Wells of each coated plate, along with uncoated controls, were aliquoted 0.50 ml each of the protein solutions. The samples were run in triplicate.

[0056] The plates were placed on a rocker table for 30 minutes at 23°C. The protein solutions were then emptied from the plates, and the wells were rinsed three times with dI H₂O. Thereafter, 1.0 ml of colloidal gold stain was added to each well (see Materials and Methods). The plates were placed on a rocker table overnight, rinsed three times with dI H₂O, and air dried. Absorbance at 550 nm was then read using the Cambridge Technologies Plate Reader.

[0057] As shown in Table 4, the non-ionic surfactants were effective in preventing protein binding for a wide variety of pH's.

Example 5

Reduced Cell Attachment

[0058] This example demonstrates that non-ionic surfactants can reduce cell attachment.

[0059] The procedures used were as follows. Polystyrene 24-well plates were coated with the non-ionic surfactants listed in Table 5 in accordance with the procedures described in Materials and Methods. Uncoated plates were used as a control. For comparison, plates with a covalently attached acrylamide coating (Corning Costar Catalog #2500) and with a stearic acid coating were also tested. The stearic acid coating was applied in the same manner as the non-ionic surfactants again using a 0.1% (w/v) solution.

[0060] The plates were gamma sterilized at 1.5 Mrad and then inoculated with 5.6 x 10⁴ MDCK wells per well in 1 ml of complete media containing 5% FBS (Fetal Bovine Serum). Incubation was carried out at 37°C in a 5% CO₂ atmosphere for two days. The plates were fixed and stained, and cell attachment was noted. Following this, the wells were stained using the colloidal gold technique to detect bound protein. Only visual observations were made.

[0061] As shown in Table 5, various of the non-ionic surfactants of the invention achieved reduced cell binding and/or reduced protein binding.

[0062] Experiments were also performed with cells suspended in serum. The cells used in these experiments were MDCK cells. The surfactants used were the same as used in Table 5. Again, reduced binding of the cells to surfaces treated with a non-ionic surfactant was observed

Example 6

Requirement for a Hydrophilic Element Which Can Extend into an Aqueous Solution

[0063] This example demonstrates that the non-ionic surfactant used to produce a low binding surface must have a hydrophilic element which can extend into an aqueous solution.

[0064] A non-ionic surfactant can have a variety of structures, including a hydrophobic segment and a hydrophilic segment attached end-to-end, a central hydrophobic segment attached on each end to a hydrophilic segment, or a central hydrophilic segment attached on each end to a hydrophobic segment. These three molecular forms can have similar HLBs, but the first two variations have at least one hydrophilic endgroup whereas the third variation, with its hydrophilic segment in the center, has hydrophobic endgroups.

[0065] The non-ionic surfactants listed in Table 6 were tested for their ability to produce a low binding surface using the techniques described in Materials and Methods. Polystyrene was used as the hydrophobic test surface and IgG/Au staining was used as the readout.

[0066] As shown in Table 6, for similar HLB numbers, surfaces coated with surfactants having at least one hydrophilic endgroup are effective in inhibiting protein binding, while surfaces coated with surfactants that do not have a hydrophilic endgroup are ineffective in inhibiting protein binding. The need for a hydrophilic element which can extend into an aqueous solution is clear from this data.

[0067] It should be noted that a surfactant suitable for use in the present invention need not have a hydrophilic endgroup but may have one or more hydrophilic groups attached to a hydrophobic backbone anywhere along the backbone's length provided such group or groups can extend into an aqueous solution.

Example 7

Drying a Surfactant-Coated Surface Is Critical to its Durability

[0068] This example demonstrates the criticality of drying a surfactant-coated surface in order to achieve a durable low binding surface. More particularly, the purpose of the experiments of this example was to determine if non-ionic surfactant molecules absorb to a polymer surface to form a durable coating during aqueous solution exposure or only after the molecules have been dried onto the surface.

[0069] Duplicate 24-well plates were prepared as follows. Individual wells were filled with 3 ml of non-ionic surfactants at 0.5% (weight/volume) in H₂O/isopropanol (90/10 weight/volume). The surfactants used are listed in Table 7

[0070] The duplicate plates were incubated for 30 minutes at room temperature on a rocker table and then emptied. One plate was tapped out and then allowed to dry at room temperature in a hood. It was then rinsed 5X with dI H₂O. The second plate was not allowed to dry, but instead was immediately rinsed 5X with H₂O. The plates were then tested for protein binding by filling each well with 0.5 ml of IgG (horse) at 10 µg/ml in PBS buffer and incubating for 30 minutes on a rocker table at room temperature. The plates were then rinsed with dI H₂O and-stained overnight with colloidal Au.

[0071] The results are shown in Table 7. As clearly demonstrated by this data, it is necessary to dry the surfactant molecules onto the plate to provide a durable coating. The data also shows the importance of using a surfactant which has a low HLB number.

Example 8

Effect of Rinsing Surfaces Exposed to Aqueous Solutions of Non-Ionic Surfactants

[0072] This example demonstrates that while the drying step is critical to durability, it is not required to achieve a low binding surface.

[0073] Medium binding (catalog # 2587) and high binding (catalog # 2581) polystyrene 96-well plates (8-well strips) manufactured by Corning Costar were used as follows. Individual wells of duplicate strips were filled with 0.3 ml of non-ionic surfactant 0.1% (weight/volume) in H₂O/isopropanol (95/5 volume/volume). The surfactants tested are listed in Table 8. Strips were incubated for 30 minutes at room temperature on a rocker table. Strips were then emptied and tapped out.

[0074] A first strip set, containing one high binding strip and one medium binding strip, was not rinsed; a second strip set, again containing one high binding strip and one medium binding strip, was rinsed 5X with dI H₂O. Immediately following this procedure, each well was aliquoted 0.10 ml of GAM-IgG-HRP (goat-anti-mouse-IgG-horseradish peroxidase enzyme labeled antibody; Kirkegaard and Perry, Gaithersburg, MD, Catalog # 074-1806) at 1.0 µg/ml in PBS buffer (pH=7.4). Strips were incubated for one hour at room temperature on a rocker table. Strips were then rinsed 5X with PBS buffer containing 0.02% Tween-20 followed by post rinsing with H₂O.

[0075] Bound antibody was measured colorimetrically using an ABTS substrate reagent kit (Kirkegaard and Perry catalog # 50-62-01). 0.10 ml of the ABTS/H₂O₂ solution was added to each well. A blue color forms as the ABTS reacts with the H₂O₂ via the peroxidase enzyme carried by the bound antibody. Absorbance at 405nm was measured on a Cambridge Technologies # 7520 Plate Reader.

[0076] Table 8 sets forth absorbance data as measured approximately 2 minutes after the ABTS/H₂O₂ solution was added to the strip. This absorbance data shows that (1) rinsing prior to drying removed the non-ionic surfactants from the strips thus preventing them from providing a low binding surface, and (2) even without drying, the surfactants achieved low binding provided they were not rinsed away.

Example 9

Determination of Coating Durability Using Water Washes

[0077] This example demonstrates the effect of HLB number on the durability of non-ionic surfactant coatings on polystyrene.

[0078] The non-ionic surfactants used in this example are set forth in Table 9. Polystyrene plates having 24 wells (Corning Costar # 9447) were coated with these surfactants as described in Materials and Methods.

[0079] Durability was tested by adding dI H₂O to the wells of a test plate (3.0 mls/well), following which the plate was placed on a rocker table. The rocker table times and temperatures, as well as the number of repetitions of the dI H₂O treatment, are set forth in Table 9. The protein binding properties of the various plates were determined using a protein solution which for each well comprised 0.5 ml of IgG (10 µg/ml) in 0.1M NaHCO₃ pH 9.4 buffer. The protein solution was incubated with the plate on a rocker table for 30 minutes at 23°C, following which the plate was H₂O rinse 3X (3 mls each time). The colloidal gold staining procedure described above was used to reveal bound protein. The staining comprised an overnight incubation on a rocker table at 23°C, a dI H₂O rinse, drying at room temperature, and reading at 550 nm.

[0080] As shown in Table 9, surfactants having HLB numbers of 1.0, 1.8, 2.0, 4.3, and 4.7 were durable, while those having HLB numbers of 6.7 and -above-were not durable.

Example 10

Determination of Coating Durability Using Protein Solution Washes

[0081] This example demonstrates the effect of HLB number on the durability of non-ionic surfactant coatings on polystyrene, polypropylene, polymethyl methacrylate, and a PVDF copolymer.

[0082] The non-ionic surfactants used in this example are set forth in Table 10. The procedures used were as follows. Injection molded assay plates (96-well format) were made from polystyrene, (STYRON Dow 685D), polypropylene (Exxon 9374), polymethyl methacrylate (ATO HAAS), and KYNAR FLEX 2800 PVDF copolymer (ATO HAAS). Each well was aliquoted 0.10 ml of IgG (Horse Standard, Pierce) at 10 µg/ml in 0.01 M PBS buffer (pH=7.4) and placed on a rocker table for 30 minutes at 23°C. The protein solution was then emptied from the wells and a fresh protein solution was aliquoted to each well and the entire procedure repeated a total of six times. The plates were then rinsed three times with dI H₂O (0.3 ml/well). Colloidal Au stain was added to each well (0.3 ml/well) and the plates were placed on a rocker table overnight. Plates were then rinsed 3 times with dI H₂O, allowed to dry, and absorbance at 550nm was measured using the Cambridge Technologies Plate Reader.

[0083] As shown in Table 10, the cutoff for a durable low binding surface is at a HLB number of 5, with non-ionic surfactants having a HLB number well above 5 showing substantial binding after the multi-wash procedure, surfactants at 6 or just below, showing a slight amount of binding, and surfactants below 5 (i.e., those having HLB numbers of 1.0, 2.0, and 4.3) showing essentially no binding. For those surfaces which did absorb protein, the Au stain was readily visible to the unaided eye. Also, the nondurable coatings often produced "splotchy" protein binding until repeated rinses removed the coating. For these coatings, high variability was observed even within one well.

[0084] Results similar to those reported in Table 10 were obtained when the same procedure was followed using repeated BSA protein rinses or just dI H₂O rinses, instead of IgG rinses.

Example 11

Coating Stability in the Presence of Excess Protein

[0085] This example demonstrates that exposure to large quantities of protein does not destroy the low binding properties of a hydrophilic surface coated with a non-ionic surfactant having a low HLB number.

[0086] The following procedures were employed in this example 24-well polystyrene plates were coated as described in Materials and Methods using 25 µl aliquots of 0.10 % (w/v) sorbitol mono-oleate in isopropanol. Coverage of the sorbitol mono-oleate was calculated at 10 µg/cm². Each well of the plates was exposed to 1.0 ml of BSA protein (Pierce) at 2 mg/ml in PBS buffer (pH=7.4). The plates were incubated on a rocker table for 30 minutes at room temperature, the wells were emptied, and the protein exposure was repeated a total of six times. The plates were then rinsed 3 times with dI H₂O and stained with the colloidal gold stain overnight. Plates were then rinsed with dI H₂O, dried, and the absorbance of each well was measured at 550 nm on the Cambridge Technologies plate reader.

[0087] As shown in Table 11, even in the presence of a large excess of protein, the low binding coating (which is believed to be physically absorbed and held only by van der Waals'forces) remained durable.

[0088] Similar experiments were carried out using Pluronic L-121 and L-122, and similar low protein binding results were obtained.

Example 12

Toxicity

[0089] In addition to effectively providing low binding surfaces, non-ionic surfactants generally have low toxicity.

[0090] Table 12 sets forth LD₅₀ values (grams/Kg of rat) for various non-ionic surfactants. For comparison, the LD₅₀ values for mineral oil, NaCl, and As₂O₃ are also included in this table. The low toxicity of non-ionic surfactants is evident from this data.

[0091] Cytotoxicity experiments were performed using MDCK cells. The non-ionic surfactants tested were Pluronic L-121, L-122, and P-123; Span 80 and 85; and Brij 30, 72 and 93. These molecules were coated onto 24-well plates (Corning Costar #9447) from 0.1%(w/v) in isopropanol. Untreated polystyrene and normal tissue culture treated polystyrene (Corning Costar #25820) 24-well plates were used as controls. All plates were incubated at 37°C for 48 hours with 2ml/well of complete DMEM media containing 10% Fetal Bovine Serum (FBS). This incubation was performed in order to extract any potentially toxic compounds from the coated surface into the cell growth serum. The FBS solutions were then transferred to tissue culture treated polystyrene and each well was inoculated with ≈ 2x10⁴ cells/well of MDCK cells, and then incubated at 37°C for 72 hours in a 5% CO₂ atmosphere. Cells were then stained with Gram Crystal Violet. All of the wells yielded confluent cell growth with normal cell morphology.

Example 13

Reduced Loss of Enzymatic Activity

[0092] This example demonstrates that an enzyme loses less activity when stored in a vessel coated with a non-ionic surfactant than in an uncoated vessel.

[0093] Corning Costar polystyrene medium binding (#2587) and high binding (#2581) microtiter plates were used in this example. Medium binding plates were coated with sorbitol mono-oleate and PEO(2) mono-oleate using the techniques described in Materials and Methods, specifically, coating with a 0.1% (w/v) solution of the surfactant in isopropanol followed by drying for 24 hours before use.

[0094] Horseradish peroxidase enzyme (HRP) was purchased from Sigma. A solution of 20 ng/ml HRP in 0.01 M PBS (pH = 7.4) was prepared. Aliquots of 0.1 ml were placed in six wells of 8-well strips of coated plates, uncoated medium binding plates, and uncoated high binding plates.

[0095] Samples were preincubated either for 0 or 90 minutes. Aliquots of 0.1 ml of a tetramethylbenzidine (TMB) peroxidase substrate system (Kirkegaard & Perry) were added to each well and absorbance at 405 nm of each well was monitored versus time using a Cambridge Technologies Inc. #7520 microplate reader.

[0096] The results were:

(1) When the TMB solution was added with a preincubation of 0 minutes, the enzyme activity of all four surfaces (i.e., the two surfactant coated surfaces, the uncoated medium binding surface, and the uncoated high binding surface) were identical within experiment error.

(2) When the enzyme was pre-incubated for 90 minutes in the uncoated high binding and medium binding plates, it lost ~60% and ~98% of its activity, respectively.

(3) When the enzyme was pre-incubated for 90 minutes in either of the coated plates, the loss in activity was essentially zero.

[0097] Similar results were observed when polypropylene plates were used instead of polystyrene plates.

[0098] It should be noted that this is a total solution assay, so that the effect being observed is not direct physical loss of protein (enzyme), but rather loss of biological activity.

Example 14

[0099] This example illustrates the use of the non-ionic surfactants of the invention to produce a membrane having a low binding surface.

[0100] The experiments were performed using 0.45 micron PVDF membranes. The membranes were coated two times by immersion using 0.1% (weight/volume) of the following non-ionic surfactants in isopropanol: sorbitol mono-oleate, Pluronic® L-121, Pluronic® L-122, and Pluronic® L-123. The membrane was dried at room temperature after each of the two immersion coatings. The coated membranes were in abated with IgG 10 µg/ml in 0.1M PBS (pH 7.4) for 30 minutes at room temperature on an orbital shaker. The membranes were then rinsed three times with water in a clean plate, each rinse being performed for 5 minutes on the shaker. Thereafter, the membranes were stained with Au overnight, again on the shaker. An uncoated PVDF membrane was used as a control.

[0101] Essentially no protein binding was seen with the Pluronic® surfactants. The sorbitol mono-oleate surfactant was found to exhibit a level of protein binding substantially identical to that of the uncoated PVDF. Although not wishing to be bound by any particular theory of operation, it is believed that the results obtained with sorbitol mono-oleate are related to the short length of this molecule's philic end (i.e., approximately 5 Å). For comparison, the Pluronic® L. :21, Pluronic® L-122, and Pluronic® L-123 surfactants have philic ends whose lengths are approximately 16, 36, and 61 Å, respectively. When coating a membrane having small pores and thus a larger surface area, it is believed that the length of a surfactant's philic ends may play a more important role in achieving a low binding surface than in other applications of the invention.

Example 15

In-Mold Coating

[0102] The in-mold coating aspects of the invention were tested by molding 24-well polystyrene plates. Sorbitol mono-oleate in isopropanol was used as the non-ionic surfactant at concentrations of 0.01% (w/v) and 0.1% (w/v). The 0.01% concentration was estimated to produce less than one monolayer of surfactant on the finished product, while the 0.1% concentration produced about 7 monolayers. The surfactant was applied to the mold using a Crown sprayer (Crown Industrial Products, Hebron, Illinois; Catalog #8011), which produces a sufficiently fine spray of droplets to uniformly coat the mold. The mold was sprayed before each part was made.

[0103] The 0.1% solution produced a finished product which was clear and exhibited essentially no protein binding when tested with IgG protein and colloidal gold staining. With the 0.01% concentration, pooling and missed areas were seen which bound protein.

[0104] Concentrations above 0.1%, i.e., concentrations ranging up to 2%, were also tested and found to work successfully.

[0105] Similar experiments were performed with a 1% isopropanol solution of glycerol mono-stearate. The part again exhibited low protein binding. A 15-25% haze, however, was observed, which made the part less transparent than an uncoated part (3-5% haze). Coating with a 0.01% isopropanol solution of sorbitol tri-stearate, on the other hand, produced a part which had the same level of haze as an uncoated part, i.e., 3-5% haze. Similar results, i.e., low hazing, were achieved with a 1% solution of ethylene glycol mono-stearate and a 1% solution of sorbitol tri-oleate. It is believed that the hazing seen with the 1% solution of glycerol mono-stearate may have been due to the use of a sprayer which did not produce a sufficiently fine spray of droplets to completely coat the mold with surfactant.

[0106] Bars of polystyrene were also coated with surfactants using the in-mold process and exhibited reduced protein binding.

[0107] Although preferred and other embodiments of the invention have been described herein, further embodiments may be perceived by those skilled in the art without departing from the scope of the invention as defined by the following claims.

Table 1

Coating	HLB No.	Non-Sterilized		Sterilized
		Total	Tight	Total	Tight
High Binding PS (uncoated)	NA	NA	NA	847	768
Medium Binding PS (uncoated)	NA	861	356	NA	NA
BSA Protein	NA	594	173	ND	ND
PEO/PPO Block Copolymer, Pluronic® L-121	1	18	4	ND	ND
PEO/PPO Block Copolymer, Pluronic® L-122	2	12	3	ND	ND
PEOIPPO Block Copolymer, Pluronic® P-123	7	7	2	ND	ND
PEOIPPO Block Copolymer, Pluronic® F-127	18	NO.	2	ND	ND
PEO(2) CETYL ETHER	5.3	18	11	54	25
PEO(4) LAURYL ETHER	9.7	2	2	10	4
PEO(2) OLEYL ETHER	4.9	7	5	22	4
PEO(4) SORBITOL MONO-LAURATE	13.3	4	2	5	3
PEO(4) SORBITOL MONO-STEARATE	9.6	37	18	130	80
PEO(20) SORBITOL TRI-STEARATE	10.5	5	2	7	4
PEO(8) STEARATE	11.1	6	3	15	9
PEO(2) STEARYL ETHER	4.9	83	28	36	17
SORBITOL MONO-OLEATE	4.3	31	7	33	5

Table 2

Coating	HLB No.	% Reduction in Protein Binding
High Binding PS - (uncoated)	NA	0.0
Medium Binding PS - (uncoated)	NA	50.0
GLYCEROL MONO-OLEATE	3.4	92.4
PEO(2) CETYL ETHER	5.3	99.9
PEO(4) LAURYL ETHER	9.7	99.3
PEO(2) OLEYL ETHER	4.9	99.9
PEO(4) SORBITOL MONO-LAURATE	13.3	99.8
PEO(4) SORBITOL MONO-STEARATE	9.6	99.5
PEO(20) SORBITOL TRI-STEARATE	10.5	99.7
PEO(8) STEARATE	11.1	99.1
PEO(2) STEARYL ETHER	4.9	97.9
SORBITOL MONO-OLEATE	4.3	92.7
SORBITOL MONO-PALMITATE	6.7	88.9*
SORBITOL MONO-STEARATE	4.7	83.1*

*The 88.9% and 83.1 % reductions for sorbitol mono-palmitate and sorbitol mono-stearate are believed to be due to incomplete coating of the surface of the plate as a result of coating defects (pinholes). Crystals were observed on the surface of the plate in these cases. Although not analyzed, the crystals are believed to be composed of the surfactant. With complete coating, these surfactants can achieve even greater reductions in binding.

Table 3

Coating	HLB No.	IgG Protein Binding Absorbance (550nm)
Medium Binding PS	NA	0.15
PEO(2) CETYL ETHER	5.3	0.00
PEO(4) LAURYL ETHER	9.7	0.00
PEO(2) OLEYL ETHER	4.9	0.00
PEO(4) SORBITOL MONO-LAURATE	13.3	0.00
PEO(4) SORBITOL MONO-STEARATE	9.6	0.00
PEO(20) SORBITOL TRI-STEARATE	10.5	0.00
PEO(8) STEARATE	11.1	0.00
PEO(2) STEARYL ETHER	4.9	0.00
SORBITOL MONO-LAURATE	8.6	0.00
SORBITOL MONO-OLEATE	4.3	0.00
SORBITOL MONO-PALMITATE	6.7	0.00
SORBITOL MONO-STEARATE	4.7	0.00

Table 4

		Absorbance (550nm)
Coating	HLB No.	pH=4.6	pH=7.4	pH=9.2
PS (uncoated)	NA	0.08	0.10	0.12
PEO(2) CETYL ETHER	5.3	0.00	0.00	0.00
PEO(4) LAURYL ETHER	9.7	0.00	0.00	0.00
PEO(2) OLEYL ETHER	4.9	0.00	0.00	0.00
PEO(4) SORBITOL MONO-STEARATE	9.6	0.00	0.00	0.00
PEO(20) SORBITOL TRI-STEARATE	10.5	0.00	0.00	0.00
PEO(8) STEARATE	11.1	0.00	0.00	0.00
PEO(2) STEARYL ETHER	4.9	0.00	0.00	0.00
SORBITOL MONO-LAURATE	8.6	0.00	0.00	0.00
SORBITOL MONO-OLEATE	4.3	0.00	0.00	0.00
SORBITOL MONO-PALMITATE	6.7	0.00	0.00	0.00
SORBITOL MONO-STEARATE	4.7	0.00	0.00	0.00

Table 5

Coating	HLB No.	MDCK Attachment	Visual Color Upon Protein Staining
PS (uncoated)	NA	Many attached and extended cells	Very Dark
Covalently attached photo-acrylamide coating (Corning Costar catalog #2500)	NA	None	ND because Au stain binds to acrylamide coating
STEARIC ACID		Many attached and extended cells	Very dark
PEO(2) CETYL ETHER	5.3	ND	None
PEO(4) LAURYL ETHER	9.7	None	None
PEO(2) OLEYL ETHER	4.9	ND	None
PEO(20) SORBITOL MONO-LAURATE	16.6	None	None
PEO/PPO Block Copolymer Pluronic® L-121	1	None	None
PEO(4) SORBITOL MONO-STEARATE	9.6	None	None
PEO(20) SORBITOL TRI-STEARATE	10.5	None	None
PEO(8) STEARATE	11.1	Coating was uneven; could not see cells	None in most areas; a few splotchy areas
SORBITOL MONO-OLEATE	4.3	Slight; coating may have been uneven	None in most areas; a few splotchy areas
SORBITOL MONO-PALMITATE	6.7	None	None
SORBITOL MONO-STEARATE	4.7	Coating was uneven; did not appear to have cells attached	None in most areas; a few splotchy areas

Table 6

Coating	HLB No.	Type of Endgroups	Bound Protein Absorbance at 550nm
PS (uncoated)	NA	Uncoated	0.10
PEO/PPO Block Copolymer, Pluronic® L-121	1	Philic (both)	0.00
PEO/PPO Block Copolymer, Pluronic® L-122	2	Philic (both)	0.00
PEO/PPO Block Copolymer, Pluronic® P-123	7	Philic (both)	0.00
PEO(2) CETYL ETHER	5.3	Philic (one)	0.00
PEO(200) DI-OLEATE	5.0	Phobic (both)	0.15
PEO(400) DI-OLEATE	8.5	Phobic (both)	0.15
PEO(400) DI-STEARATE	8.8	Phobic (both)	0.10
PEO(600) DI-STEARATE	10.6	Phobic (both)	0.10
PEO(2) OLEYL ETHER	4.9	Philic (one)	0.00
PEO(4) SORBITOL MONO-LAURATE	13.3	Philic (one)	0.00
PEO(20) SORBITOL TRI-STEARATE	10.5	Philic (one)	0.00
POLYPROPYLENE OXIDE (4,000 M.W.)	<0.5	Philic (two)	0.00
SORBITOL MONO-OLEATE	4.3	Philic (one)	0.00

Table 7

Coating	HLB No.	Absorbance (550nm) Dried before H2O rinse	Absorbance (550nm) H2O rinsed before drying
PS (uncoated)	NA	0.13-0.15	0.13-0.15
PEO/PPO Block Copolymer, Pluronic® L-121	1	0.00	0.13-0.16
PEO/PPO Block Copolymer, Pluronic® L-122	2	0.00	0.12-0.15
PEO/PPO Block Copolymer, Pluronic® P-123	7	0.08-0.11	0.14-0.16
PEO/PPO, Block Copolymer, Pluronic® F-127	18	0.13-0.16	0.13-0.16
PEO(4) LAURYL ETHER	9.7	0.08-0.10	0.13-0.16
PEO(2) OLEYL ETHER	4.9	0.00	0.14-0.16
PEO(20) SORBITOL MONO-LAURATE	16.6	0.14-0.16	0.13-0.16

Table 8

		Absorbance (405 nm)
Surfactant	HLB No.	High Binding Polystyrene		Medium Binding Polystyrene
		No Rinse	Rinsed	No Rinse	Rinsed
None	NA	2.00	2.10	1.90	1.90
PEO/PPO Block Copolymer, Pluronic® L-121	1	0.00	1.90	0.00	1.00
PEO/PPO Block Copolymer, Pluronic® L-122	2	0.00	1.90	0.00	1.70
PEO/PPO Block Copolymer, Pluronic® P-123	7	0.00	1.80	0.00	1.70
PEO/PPO Block Copolymer, Pluronic® F-127	18	0.00	1.00	0.00	1.40
PEO(4) LAURYL ETHER	9.7	ND	2.10	ND	1.90
PEO(2) OLEYL ETHER	4.9	ND	2.10	ND	1.90
PEO(20) SORBITOL MONO-LAURATE	16.6	0.00	2.00	0.00	1.90

Table 9

Coating	HLB No.	Absorbance (550 nm)
		Initial	1 Time. 1 hr 23°C	1 Time 24 hr 23°C	1. Time 24 hr 37°C	1 Time 24 hr 70°C	6 Times 30 min each 23°C
PS (uncoated)	NA	0.10	0.10	0.10	0.10	0.10	0.10
PEO/PPO Block Copolymer, Pluronic® L-121	1	0.00	0.00	0.00	0.00	0.00	0.00
PEO/PPO Block Copolymer, Pluronic® L-122	2	0.00	0.00	0.00	0.00	0.00	0.00
PEO/PPO Block Copolymer, Pluronic® P-123	7	0.00	0.00	0.00	0.00	0.00-0.03	0.02-0.06
PEO/PPO Block Copolymer, Pluronic® F-127	18	0.00	0.05-0.10	0.10	0.10	0.10	0.10
PEO(4) SORBITOL MONO-LAURATE	13.3	0.00	0.09	0.10	0.10	0.10	0.19
PEO(20) SORBITOL MONO-LAURATE	16.6	0.00	0.02	0.05	0.10	0.10	0.10
PEO(4) SORBITOL MONO-STEARATE	9.6	0.00	0.00	0.02	0.10	0.10	0.10
PEO(20) SORBITOL TRI-STEARATE	10.5	0.00	0.00	0.02	0.10	0.10	0.10
SORBITOL MONO-LAURATE	8.6	0.00	0.00	0.00	0.00	0.00-0.02	0.02-0.06
SORBITOL MONO-OLEATE	4.3	0.00	0.00	0.00	0.00	0.00	0.00
SORBITOL MONO-PALMITATE	6.7	0.00	0.00	0.00	0.00	0.00	0.02-0.06
SORBITOL MONO-STEARATE	4.7	0.00	0.00	0.00	0.00	0.00	0.00
SORBITOL TRI-OLEATE	1.8	0.00	0.00	0.00	0.00	0.00	0.00

Table 10

Coating	HLB No.	Absorbance (550nm)
		Polystyrene	Polypropylene	Polymethyl Methacrylate	KYNAR® Flex 2800 PVDF copolymer
As Molded Not Coated	NA	0.10	0.08	0.15	0.15
PEO/PPO Block Copolymer, Pluronic® L-31	6-7	0.0-0.2	0.04	0.02-0.04	0.04-0.12
PEO/PPO Block Copolymer, Pluronic® L-62	5-6	ND	0.04	0.02	0.02-0.08
PEO/PPO Block Copolymer, Pluronic® L-64	15	ND	0.08	0.02	0.02-0.08
PEO/PPO Block Copolymer, Pluronic® L-121	1	0.00	0.00	0.00	0.00
PEO/PPO Block Copolymer, Pluronic® L-122	2	0.00	0.00	0.00	0.00
PEO/PPO Block Copolymer, Pluronic® P-123	7	0.02-0.04	0.03	0.01	0.02-0.08
PEO/PPO, Block Copolymer, Pluronic® F-127	18	0.07-0.10	0.04-0.08	0.02-0.08	0.06
SORBITOL MONO-OLEATE	4.3	0.00	0.00	ND	ND

Table 11

Number of Exposures	Total Exposure Time (min)	Ratio of Weight of Protein to Weight of Coating	Absorbance at 550 nm	Absorbance of Uncoated Polystyrene Control
1	30	80	0.0	0.10
2	60	160	0.0	0.10
3	90	240	0.0	0.10
4	120	320	0.0	0.10
5	150	400	0.0	0.10
6	180	480	0.0	0.10

Table 12

Molecule	HLB No.	LD50 grams/Kg (rat)
PEO(4) LAURYL ETHER	9.7	>5
PEO(4) SORBITOL MONO-LAURATE	13.3	>38
PEO(20) SORBITOL TRI-STEARATE	10.5	>39
PEO(8) STEARATE	11.1	>31
PEO(2) STEARYL ETHER	4.9	>25
SORBITOL MONO-OLEATE	4.3	>39
SORBITOL MONO-STEARATE	4.7	>15
SORBITOL TRI-STEARATE	2.1	>15
MINERAL OIL	NA	22
NaCl	NA	4
As2O3	NA	0.02

Table 13

Molecule	HLB No.	Tradename
ETHYLENE GLYCOL MONO-STEARATE	3.0	Emerest® 2350
GLYCEROL MONO-OLEATE	3.4	Emerest® 2421
GLYCEROL MONO-STEARATE	3.4	Emerest® 2400
PEO/PPO Block Copolymer	6-7	Pluronic® L-31
PEO/PPO Block Copolymer	4	Pluronic® L-61
PEO/PPO Block Copolymer	5	PluroniC® L-62
PEO/PPO Block Copolymer	15	Pluronic® L-64
PEO/PPO Block Copolymer	1	Pluronic® L-121
PEO/PPO Block Copolymer	2	Pluronic® L-122
PEO/PPO Block Copolymer	7	Pluronic®P-123
PEO/PPO Block Copolymer	18	Pluronic® F-127
PEO(2) CETYL ETHER	5.3	Brij® 52.
PEO(200) DI-OLEATE	5.0	Maypeg® 200 DO
PEO(400) DI-OLEATE	8.5	Maypeg® 400 DO
PEO(400) DI-STEARATE	8.8	Maypeg® 400 DS
PEO(600) DI-STEARATE	10.6	Maypeg® 400 DS
PEO(4) LAURYL ETHER	9.7	Brij® 30
PEO(200) MONO-OLEATE	8.3	Emerest® 2624
PEO(2) OLEYL ETHER	4.9	Brij® 93
PEO(4) SORBITOL MONO-LAURATE	13.3	Tween® 21
PEO(20) SORBITOL MONO-LAURATE	16.6	Tween® 20
PEO(4) SORBITOL MONO-STEARATE	9.6	Tween® 61
PEO(20) SORBITOL TRI-STEARATE	10.5	Tween® 65
PEO(8) STEARATE	11.1	Myrj® 45
PEO(2) STEARYL ETHER	4.9	Brij® 72
POLYPROPYLENE OXIDE, (4,000 M.W.)	<0.5	Purchased from Aldrich, Milwaukee,
SORBITOL MONO-LAURATE	8.6	SPAN®20
SORBITOL MONO-OLEATE	4.3	SPAN® 80
SORBITOL MONO-PALMITATE	6.7	SPAN® 40
SORBITOL MONO-STEARATE	4.7	SPAN® 60
SORBITOL TRI-OLEATE	1.8	SPAN® 85
SORBITOL TRI-STEARATE	2.1	SPAN® 65

[0108] Brij® is a registered trademark of ICI Americas, Wilmington, DE. Everest® is a registered trademark of Henkel Corp., Cincinnati, OH Maypeg® is a registered trademark of PPG Industries, Gurnee, IL Myrj® is a registered trademark of ICI Americas, Wilmington, DE. Pluronic® is a registered trademark of BASF, Parsippan NJ. Span® is a registered trademark of ICI Americas, Wilmington, DE. Tween® is a registered trademark of ICI Americas, Wilmington, DE.

Claims

1. A method for making a body composed of a hydrophobic polymer, said method comprising:

(a) providing a mold for forming the body;

(b) applying a non-ionic surfactant to a surface of the mold, said non-ionic surfactant having (i) a hydrophilic-lipophilic balance number which is less than or equal to 5 and (ii) at least one hydrophilic element which can extend into an aqueous solution; and

(c) using the mold to form the body with at least part of the body being formed by contact with the surface to which the non-ionic surfactant was applied so that at least some non-ionic surfactant is transferred from the surface to said part of the body.

2. The method of Claim 1 comprising the additional step after step (c) of contacting the body with an aqueous solution containing an organic material, said body exhibiting reduced binding of the organic material in comparison to a body formed using a mold to which the non-ionic surfactant of step (b) has not been applied.

3. The method of Claim 2 wherein the body exhibits a level of binding of the organic material which is less than 0.5 times the level of binding exhibited by a body formed using a mold to which the non-ionic surfactant of step (b) has not been applied.

4. The method of Claim 2 wherein the body exhibits a level of binding of the organic material which is less than 0.3 times the level of binding exhibited by a body formed using a mold to which the non-ionic surfactant of step (b) has not been applied.

5. The method of Claim 1 comprising the additional step after step (c) of contacting the body with an aqueous solution containing an organic material for a period of time, said organic material having biological activity and exhibiting enhanced retention of its biological activity over said period of time when the aqueous solution is in contact with the body than when the aqueous solution is in contact with a body formed using a mold to which the non-ionic surfactant of step (b) has not been applied.

6. The method of Claim 5 wherein the period of time is 90 minutes and the organic material exhibits a retention of biological activity when the aqueous solution is in contact with the body which is at least 1.5 times the retention of biological activity exhibited when the aqueous solution is in contact with a body formed using a mold to which the non-ionic surfactant of step (b) has not been applied.

7. The method of Claim 1 wherein the non-ionic surfactant has a hydrophilic-lipophilic balance number which is less than or equal to 2.5.

8. The method of Claim 7 wherein the non-ionic surfactant comprises an ethylene oxide/propylene oxide block co-polymer.

9. The method of Claim 7 wherein the non-ionic surfactant comprises polypropylene oxide.

10. The method of Claim 1 wherein the hydrophobic polymer comprises polystyrene.

11. The method of Claim 1 wherein the non-ionic surfactant is applied to the mold in the form of a solution of the non-ionic surfactant and a solvent.

12. The method of Claim 11 wherein the solvent comprises an alcohol.

13. A body made by the method of Claim 1.

14. The body of Claim 13 wherein the body comprises labware.

Ansprüche

1. Verfahren zur Herstellung eines Körpers, zusammengesetzt aus einem hydrophoben Polymer, wobei das Verfahren umfasst:

(a) Bereitstellen einer Form zum Ausbilden des Körpers;

(b) Aufbringen eines nicht-ionischen, oberflächenaktiven Stoffes auf eine Oberfläche der Form, wobei der nicht-ionische, oberflächenaktive Stoff (i) eine hydrophile-lipophile Gleichgewichtszahl, die kleiner oder gleich 5 ist, und (ii) mindestens ein hydrophiles Element, das sich in eine wässrige Lösung erstrecken kann, besitzt; und

(c) Verwenden der Form, um den Körper auszubilden, wobei mindestens ein Teil des Körpers durch Kontakt mit der Oberfläche, auf welche der nicht-ionische, oberflächenaktive Stoff aufgebracht wurde, ausgebildet wird, so dass wenigstens etwas nicht-ionischer, oberflächenaktiver Stoff von der Oberfläche auf den Teil des Körpers übertragen wird.

2. Verfahren gemäß Anspruch 1, das den zusätzlichen Schritt nach Schritt (c) des Inkontaktbringen des Körpers mit einer wässrigen Lösung, die ein organisches Material enthält, umfasst, wobei der Körper eine verminderte Bindung des organischen Materials im Vergleich mit einem Körper, der unter Verwendung einer Form ausgebildet wurde, auf die der nicht-ionische, oberflächenaktive Stoff aus Schritt (b) nicht aufgebracht wurde, zeigt.

3. Verfahren gemäß Anspruch 2, wobei der Körper einen Grad der Bindung des organischen Materials zeigt, welcher weniger als das 0,5-fache des Grades der Bindung ist, der von einem Körper gezeigt wird, der unter Verwendung einer Form, auf welche der nicht-ionische, oberflächenaktive Stoff aus Schritt (b) nicht aufgebracht wurde, ausgebildet wurde.

4. Verfahren gemäß Anspruch 2, wobei der Körper einen Grad der Bindung des organischen Materials zeigt, welcher weniger als das 0,3-fache des Grades der Bindung ist, der von einem Körper gezeigt wird, der unter Verwendung einer Form, auf welche der nicht-ionische, oberflächenaktive Stoff aus Schritt (b) nicht aufgebracht wurde, ausgebildet wurde.

5. Verfahren gemäß Anspruch 1, das den zusätzlichen Schritt nach Schritt (c) des Inkontaktbringens des Körpers mit einer wässrigen Lösung, die ein organisches Material enthält, über einen Zeitraum umfasst, wobei das organische Material biologische Aktivität besitzt und eine verbesserte Beibehaltung seiner biologischen Aktivität über den Zeitraum zeigt, wenn die wässrige Lösung in Kontakt mit dem Körper ist, als wenn die wässrige Lösung in Kontakt mit einem Körper ist, der unter Verwendung einer Form, auf welche der nicht-ionische, oberflächenaktive Stoff aus Schritt (b) nicht aufgebracht wurde, ausgebildet wurde.

6. Verfahren gemäß Anspruch 5, wobei der Zeitraum 90 Minuten beträgt und das organische Material eine Beibehaltung der biologischen Aktivität zeigt, wenn die wässrige Lösung in Kontakt mit dem Körper ist, welche mindestens das 1,5-fache der Beibehaltung der biologischen Aktivität beträgt, die gezeigt wird, wenn die wässrige Lösung in Kontakt mit einem Körper ist, der unter Verwendung einer Form, auf welche der nicht-ionische, oberflächenaktive Stoff aus Schritt (b) nicht aufgebracht wurde, ausgebildet wurde.

7. Verfahren gemäß Anspruch 1, wobei der nicht-ionische, oberflächenaktive Stoff eine hydrophile-lipophile Gleichgewichtszahl besitzt, welche weniger als oder gleich 2,5 beträgt.

8. Verfahren gemäß Anspruch 7, wobei der nicht-ionische, oberflächenaktive Stoff ein Ethylenoxid/Propylenoxid-Blockcopolymer umfasst.

9. Verfahren gemäß Anspruch 7, wobei der nicht-ionische, oberflächenaktive Stoff Polypropylenoxid umfasst.

10. Verfahren gemäß Anspruch 1, wobei das hydrophobe Polymer Polystyrol umfasst.

11. Verfahren gemäß Anspruch 1, wobei der nicht-ionische, oberflächenaktive Stoff auf die Form in Form einer Lösung des nicht-ionischen, oberflächenaktiven Stoffes und eines Lösungsmittels aufgebracht wird.

12. Verfahren gemäß Anspruch 11, wobei das Lösungsmittel einen Alkohol umfasst.

13. Körper, der durch das Verfahren gemäß Anspruch 1 hergestellt wurde.

14. Körper gemäß Anspruch 13, wobei der Körper eine Laborware umfasst.

Revendications

1. Procédé de fabrication d'un corps, comprenant un polymère hydrophobe, ledit procédé comprenant :

(a) la fourniture d'un moule pour former le corps ;

(b) l'application d'un tensioactif non ionique sur une surface du moule, ledit tensioactif non ionique ayant (i) un indice d'équilibre hydrophile - lipophile qui est inférieur ou égal à 5 et (ii) au moins un élément hydrophile qui peut s'expanser dans une solution aqueuse ; et

(c) l'utilisation du moule pour former le corps avec au moins une partie du corps qui est formée par contact avec la surface sur laquelle le tensioactif non ionique a été appliqué de sorte qu'au moins un peu de tensioactif non ionique soit transféré de la surface à ladite partie du corps.

2. Procédé selon la revendication 1, comprenant l'étape additionnelle après l'étape (c) de mise en contact du corps avec une solution aqueuse contenant une matière organique, ledit corps présentant une liaison réduite du matériau organique en comparaison avec un corps formé en utilisant un moule sur lequel le tensioactif non ionique de l'étape (b) n'a pas été appliqué.

3. Procédé selon la revendication 2, dans lequel le corps présente un taux de liaison de la matière organique qui est inférieur à 0,5 fois le taux de liaison présenté par un corps formé en utilisant un moule sur lequel le tensioactif non ionique de l'étape (b) n'a pas été appliqué.

4. Procédé selon la revendication 2, dans lequel le corps présente un taux de liaison de la matière organique qui est inférieur à 0,3 fois le taux de liaison présenté par un corps formé en utilisant un moule sur lequel le tensioactif non ionique de l'étape (b) n'a pas été appliqué.

5. Procédé selon la revendication 1, comprenant l'étape additionnelle après l'étape (c) de mise en contact du corps avec une solution aqueuse contenant une matière organique pendant une certaine durée, ladite matière organique ayant une activité biologique et présentant une rétention accrue de son activité biologique pendant ladite durée quand la solution aqueuse est en contact avec ce corps plutôt que quand la solution aqueuse est en contact avec un corps formé en utilisant un moule sur lequel le tensioactif non ionique de l'étape (b) n'a pas été appliqué.

6. Procédé selon la revendication 5, dans lequel la durée est de 90 minutes et la matière organique présente une rétention de l'activité biologique quand la solution aqueuse est en contact avec ce corps qui est d'au moins 1,5 fois la rétention de l'activité biologique présentée quand la solution aqueuse est en contact avec un corps formé en utilisant un moule sur lequel le tensioactif non ionique de l'étape (b) n'a pas été appliqué.

7. Procédé selon la revendication 1, dans lequel le tensioactif non ionique a un indice d'équilibre hydrophile - lipophile qui est inférieur ou égal à 2,5.

8. Procédé selon la revendication 7, dans lequel le tensioactif non ionique comprend un copolymère à bloc d'oxyde d'éthylène/oxyde de propylène.

9. Procédé selon la revendication 7, dans lequel le tensioactif non ionique comprend de l'oxyde de polypropylène.

10. Procédé selon la revendication 1, dans lequel le polymère hydrophobe comprend du polystyrène.

11. Procédé selon la revendication 1, dans lequel le tensioactif non ionique est appliqué sur le moule sous la forme d'une solution de tensioactif non ionique et d'un solvant.

12. Procédé selon la revendication 11, dans lequel le solvant comprend un alcool.

13. Corps fabriqué par le procédé selon la revendication 1.

14. Corps selon la revendication 13, dans lequel le corps comprend du matériel de laboratoire.